Development of a novel live attenuated African Swine Fever vaccine based in the deletion of gene I177L

ABSTRACT

Provided herein are details on the construction of a recombinant African Swine Fever Virus (ASFV) live attenuated vaccine for prevention of ASF caused by various strains of ASFV, such as the highly virulent Georgia 2007 isolate (“ASFV-G”). An exemplary vaccine comprises the ASFV-GΔI1771 modified virus, a recombinant ASFV-G modified by deleting a portion of the I177L ORF rendering the I177L gene nonfunctional.

BACKGROUND OF THE INVENTION Field of Invention

The present disclosure provides details on the construction of arecombinant African Swine Fever Virus (ASFV) live attenuated vaccine forprevention of ASF caused by various strains of ASFV, such as the highlyvirulent Georgia 2007 isolate (“ASFV-G”). An exemplary vaccine comprisesthe ASFV-GΔI1771 modified virus, a recombinant ASFV-G modified bydeleting a portion of the I177L ORF rendering the I177L genenonfunctional.

Background

African Swine Fever (ASF) is a contagious viral disease of swine. Thecausative agent, ASF virus (ASFV), is a large enveloped virus containinga double-stranded DNA genome of approximately 190 kilobase pairs. ASFVshares aspects of genome structure and replication strategy with otherlarge double-stranded DNA viruses, including the Poxviridae,Iridoviridae and Phycodnaviridae (Costard et al, Phil. Trans. Royal Soc.B, (2009) 364:2683-96). ASFV infections in domestic pigs are often fataland are characterized by fever, hemorrhages, ataxia and severedepression. However, the course of infection varies, ranging from highlylethal to sub-clinical, depending on the host characteristics and theparticular virus strain (Tulman et al, Curr. Top. Microbial. Immunol.(2009) 328:43-87).

Currently, the disease is endemic in more than twenty sub-SaharanAfrican countries. In Europe, ASF is still endemic on the island ofSardinia (Italy) and new outbreaks have been declared in the Caucasusregion since 2007, affecting Georgia, Armenia, Azerbaijan and Russia.Outbreaks have been recently reported in Ukraine, Belarus, Lithuania,Latvia and Poland, affecting both wild boar and swine farms. In2018-2019 ASF spread into China, Mongolia, Vietnam, Cambodia and NorthKorea, in both wild boar and domestic swine farms. In 2019 ASF has alsospread to wild boar populations in Belgium, where ASF is currently onlyaffecting a small containment area in the country. Recent ASF outbreakspose the risk of further dissemination into neighboring countries. Theparental epidemic virus ASFV Georgia 2007/1, is a highly virulentisolate belonging to the genotype II (Chapman et al, Emerging Infect.Dis. (2011) 17:599-605), and is responsible for all the currentoutbreaks in Asia and Europe, with outbreak viruses having 90% orgreater similarity to the parental strain.

Currently, there is no commercial vaccine available for ASF and diseaseoutbreaks are controlled by animal quarantine and slaughter. Attempts tovaccinate animals using infected cell extracts, supernatants of infectedpig peripheral blood leukocytes, purified and inactivated virions,infected glutaraldehyde-fixed macrophages, or detergent-treated infectedalveolar macrophages failed to induce protective immunity (Coggins, L.,Prag. Med. Viral. (1974) 18:48-63; Forman et al, Arch. Viral., (1982)74:91-100; Kihm et al, (1987) In: African Swine Fever, Becker, Y. (ed),Martinus Nijhoff, Boston, pp 127-44; Mebus, C. A., Adv. Virus Res.,(1988) 35:251-69). Homologous protective immunity does develop in pigssurviving viral infection. Pigs surviving acute infection withmoderately virulent or attenuated variants of ASFV develop long-termresistance to homologous, but rarely to heterologous, virus challenge(Hamdy and Dardiri, Am. J. Vet. Res. (1984) 45:711-14; Ruiz-Gonzalvo etal, (1981) In: FAO/CEC Expert Consultation in ASF Research, Wilkinson,P. J. (ed), Rome, pp 206-16). Herein, we report the development of arecombinant vaccine in which a portion of the I177L gene has beendeleted from the ASFV-G genome. Vaccination of pigs with this virusprotected swine from developing ASF. Because there are not ASFV vaccinescurrently available, the development of any vaccine that may induceprotection against the lethal presentation of the disease is of greatinterest.

SUMMARY OF THE INVENTION

The present disclosure provides a genetically modified virus, whereinthe virus comprises a viral genome at least 99% identical to SEQ ID NO:2. In a particular embodiment, the viral genome comprises SEQ ID NO:2.

Also provided herein is vaccine composition against African Swine FeverVirus (ASFV), comprising a genetically modified virus comprising a viralgenome at least 99% identical to SEQ ID NO: 2. In some embodiments, theASFV strain is the ASFV-Georgia 2007 isolate.

Further provided herein is a method for the protection of swine againstASFV, comprising administering to a swine a live attenuated vaccinecomprising a genetically modified virus, wherein the virus comprises aviral genome at least 99% identical to SEQ ID NO: 2 in an amounteffective to protect the swine from clinical ASFV disease. In someembodiments, the ASFV is ASFV-G. In particular embodiments, the amounteffective to protect the swine from clinical ASFV disease is a vaccinecomprising 10²-10⁶ HAD₅₀ of the genetically modified virus.

An additional embodiment provided herein is a recombinant ASFV mutantvirus, comprising a synthetic mutation in the I177L open reading frameor in a regulatory element controlling expression of the I177L protein,resulting in a non-functional genomic I177L gene. In particularembodiments, the synthetic mutation is a deletion mutation resulting thedeletion of one or more nucleotides between positions 174471 and 175004of SEQ ID NO:1. In other embodiments, the synthetic mutation is aframeshift mutation, insertion mutation, nonsense mutation of one ormore nucleotides between positions 174471 and 175004 of SEQ ID NO:1. Insome embodiments, the mutant ASFV is an ASFV-Georgia isolate. Inspecific embodiments, the mutant ASFV comprises a genome at least 95%identical to, or at least 99% identical to SEQ ID NO: 2.

Further provided herein is a vaccine composition against ASFV-G,comprising a recombinant ASFV mutant virus, comprising a syntheticmutation in the I177L open reading frame or in a regulatory elementcontrolling expression of the I177L protein, resulting in anon-functional genomic I177L gene.

Also provided herein is a method for the protection of swine againstASFV, comprising administering to a swine a live attenuated vaccinecomprising a recombinant ASFV mutant virus, comprising a syntheticmutation in the I177L open reading frame or in a regulatory elementcontrolling expression of the I177L protein, resulting in anon-functional genomic I177L gene in an amount effective to protect saidswine from clinical ASFV disease. In particular embodiments, the ASFV isASFV-G. In some embodiments, the amount effective to protect the swinefrom clinical ASFV disease is a vaccine comprising 10²-10⁶ HAD₅₀ of thegenetically modified virus.

INCORPORATION BY REFERENCE

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The novel features of the invention are set forth with particularity inthe claims. Features and advantages of the present invention arereferred to in the following detailed description, and the accompanyingdrawings of which:

FIG. 1 provides a graphic representation of the cassette used to createthe ASFV-G ΔI177L recombinant mutant virus.

FIG. 2 provides graphic representation of in vitro growthcharacteristics of ASFV-G-ΔI177L and parental ASFV-G. Primary swinemacrophage cell cultures were infected (MOI=0.01) with each of theviruses and virus yield titrated at the indicated times post-infection.Data represent means from three independent experiments. Sensitivity ofvirus detection: ≥1.8 log₁₀ HAD₅₀/ml.

FIG. 3 provides graphic representation of viremia titers detected inpigs IM inoculated with either 10² HAD₅₀ of ASFV-G-ΔI177L or 10² HAD₅₀of ASFV-G. Each curve represents values from individual animals in eachof the group. Sensitivity of virus detection: ≥log₁₀ 1.8 log₁₀TCID₅₀/ml.

FIG. 4 provides graphic representation of kinetics of body temperaturevalues in pigs IM inoculated with either 10², 10⁴, or 10⁶ HAD₅₀ ofASFV-G-ΔI177L (filled symbols), mock inoculated (sentinels, showed inred) or 10² HAD₅₀ of ASFV-G (empty symbols) (panels on the left) andafter the challenge with 10² HAD₅₀ of ASFV-G (panels on the right). Eachcurve represents individual animal's values in each of the group.

FIG. 5 provides graphic representation of viremia titers detected inpigs IM inoculated with either 10², 10⁴, or 10⁶ HAD₅₀ of ASFV-G-ΔI177Lor 10² HAD₅₀ of ASFV-G. Viremia after the challenge with 10² HAD₅₀ ofASFV-G Each curve represents values from individual animals in each ofthe group. Sensitivity of virus detection: ≥log₁₀ 1.8 TCID₅₀/ml.

FIG. 6 provides graphic representation of anti-ASFV antibody (IgMmediated shown in panels in the left column, and IgG mediated shown inpanels in the right column) titers detected by ELISA in pigs IMinoculated with either 10², 10⁴, or 10⁶ HAD₅₀ of ASFV-G-ΔI177L. Antibodyresponse mediated by IgM Each curve represents values from individualanimals in each of the group.

DETAILED DESCRIPTION OF THE INVENTION

African swine fever virus (ASFV) is the etiological agent of acontagious and often lethal viral disease of domestic pigs that hassignificant economic consequences for the swine industry. The control ofAfrican Swine Fever (ASF) has been hampered by the unavailability ofvaccines. Experimental vaccines have been previously reported that werederived from naturally occurring, cell culture-adapted, or geneticallymodified live attenuated ASFV. However, none of these vaccines have beendeveloped for commercial use. Here we report the discovery that deletionof a previously uncharacterized gene, I177L, from the highly virulentASFV isolate Georgia isolate (ASFV-G) produces its complete attenuationin swine. Animals inoculated with the virus lacking a functional I177Lgene—such as the specific ASFV-G-ΔI177L mutant describedherein—administered intramuscularly (IM) remain clinically normal duringa 28-day observational period. Importantly, ASFV-G-ΔI177L infectedanimals were protected when challenged with the virulent parental strainASFV-G.

Preferred embodiments of the present invention are shown and describedherein. It will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions will occur to those skilled in the artwithout departing from the invention. Various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is intended that the included claims definethe scope of the invention and that methods and structures within thescope of these claims and their equivalents are covered thereby.

Technical and scientific terms used herein have the meanings commonlyunderstood by one of ordinary skill in the art to which the instantinvention pertains, unless otherwise defined. Reference is made hereinto various materials and methodologies known to those of skill in theart. Standard reference works setting forth the general principles ofrecombinant DNA technology include Sambrook et al., “Molecular Cloning:A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y., 1989; Kaufman et al., eds., “Handbook of Molecular andCellular Methods in Biology and Medicine”, CRC Press, Boca Raton, 1995;and McPherson, ed., “Directed Mutagenesis: A Practical Approach”, IRLPress, Oxford, 1991.

Any suitable materials and/or methods known to those of skill can beutilized in carrying out the instant invention. Materials and/or methodsfor practicing the instant invention are described. Materials, reagentsand the like to which reference is made in the following description andexamples are obtainable from commercial sources, unless otherwise noted.This invention teaches methods and describes tools for producinggenetically altered strains of ASFV.

As used in the specification and claims, use of the singular “a”, “an”,and “the” include plural references unless the context clearly dictatesotherwise.

The terms isolated, purified, or biologically pure as used herein, referto material that is substantially or essentially free from componentsthat normally accompany the referenced material in its native state.

The term “about” is defined as plus or minus ten percent of a recitedvalue. For example, about 1.0 g means 0.9 g to 1.1 g and all valueswithin that range, whether specifically stated or not.

The term “a nucleic acid consisting essentially of”, and grammaticalvariations thereof, means nucleic acids that differ from a referencenucleic acid sequence by 20 or fewer nucleic acid residues and alsoperform the function of the reference nucleic acid sequence. Suchvariants include sequences which are shorter or longer than thereference nucleic acid sequence, have different residues at particularpositions, or a combination thereof.

The term “adjuvant” means a substance or vehicle that non-specificallyenhances the immune response to an antigen. Adjuvants can include asuspension of minerals (alum, aluminum hydroxide, or phosphate) on whichantigen is adsorbed; or water-in-oil emulsion in which antigen solutionis emulsified in mineral oil (for example, Freund's incompleteadjuvant), sometimes with the inclusion of killed mycobacteria (Freund'scomplete adjuvant) to further enhance antigenicity. Immunostimulatoryoligonucleotides can also be used as adjuvants (for example, see U.S.Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116;6,339,068; 6,406,705; and 6,429,199). Adjuvants also include biologicalmolecules, such as costimulatory molecules.

The term “administer”/“administration” means any method of providing asubject with a substance, such as a therapeutic agent by any effectiveroute. Exemplary routes of administration include, but are not limitedto, injection (such as subcutaneous, intramuscular, intradermal,intraperitoneal, and intravenous), oral, intraductal, sublingual,rectal, transdermal, intranasal, vaginal and inhalation routes.

The terms “coding sequence” and “coding region” as used herein refer tonucleotide sequences and nucleic acid sequences, including both RNA andDNA, that encode genetic information for the synthesis of an RNA, aprotein, or any portion of an RNA or protein.

The term “effective amount” of a composition provided herein refers tothe amount of the composition capable of performing the specifiedfunction for which an effective amount is expressed. The exact amountrequired can vary from composition to composition and from function tofunction, depending on recognized variables such as the compositions andprocesses involved. An effective amount can be delivered in one or moreapplications. Thus, it is not possible to specify an exact amount,however, an appropriate “effective amount” can be determined by theskilled artisan via routine experimentation.

The terms “I177L”, “ASFV I177L”, and “genomic I177L” are synonyms andrefer to the gene defined herein as SEQ ID NO: 3, or any version of SEQID NO: 3 with base substitutions that result in a protein with asequence identical to SEQ ID NO: 4). These terms, in the appropriatecontext, can also refer to modified versions of these SEQ ID NOs, suchas those comprising deletions, insertions, and other recombinantmodifications. ASFV-G open reading frame I177L encodes a 177 amino acidprotein (SEQ ID NO: 4) and is positioned on the reverse strand betweennucleotide positions 174471 and 175004 of SEQ ID NO:1.

In the context of the present invention, the term “non-functionalgenomic I177L” refers to a modified I177L gene, located in the genome ofan ASFV, wherein such modification of the ASFV I177L gene results in noASFV I177L gene product at all or a biologically non-functional ASFVI177L gene product as compared to an unmodified functional ASFV I177Lgene. Such modifications can include, but are not limited to, full orpartial deletion of the coding sequence, disruption of the open readingframe (e.g., by insertion of a shift mutation or insertion of a nonsensecodon), modification of upstream or downstream regulatory elements,and/or any other currently known or conceivable method of inactivatingor knocking-out functional expression of such ASFV I117L gene.

The term “immunize” means to render a subject protected from aninfectious disease, such as by vaccination.

For the purpose of this invention, the “sequence identity” of tworelated nucleotide or amino acid sequences, expressed as a percentage,refers to the number of positions in the two optimally aligned sequenceswhich have identical residues (×100) divided by the number of positionscompared. A gap, i.e., a position in an alignment where a residue ispresent in one sequence but not in the other is regarded as a positionwith non-identical residues. The alignment of the two sequences isperformed by the Needleman and Wunsch algorithm (Needleman and Wunsch, JMol Biol, (1970) 48:3, 443-53). A computer-assisted sequence alignmentcan be conveniently performed using a standard software program such asGAP which is part of the Wisconsin Package Version 10.1 (GeneticsComputer Group, Madison, Wis., USA) using the default scoring matrixwith a gap creation penalty of 50 and a gap extension penalty of 3.

The phrase “high percent identical” or “high percent identity”, andgrammatical variations thereof in the context of two polynucleotides orpolypeptides, refers to two or more sequences or sub-sequences that haveat least about 80%, identity, at least about 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% nucleotide or amino acid identity, when compared and aligned formaximum correspondence, as measured using a sequence comparisonalgorithm or by visual inspection. In one exemplary embodiment, thesequences are high percent identical over the entire length of thepolynucleotide or polypeptide sequences.

The term “swine” can generally refer to any member of the Suidae familyand includes domesticated and wild pigs, hogs and boars.

A “vaccine” is herein defined as a biological agent capable of providinga protective response in an animal to which it has been delivered butnot capable of causing a serious disease. Administration of the vaccineresults in immunity from the disease. Thus, the vaccine stimulatesantibody production or cellular immunity against the disease-causingpathogen (e.g., ASFV). Immunity is herein defined as the induction ofsignificantly higher levels of protection against lethality and clinicalsymptoms following vaccination in a swine population, as compared to thenon-vaccinated group. In particular, the vaccine according to theinvention protects most of the vaccinated animals against thedevelopment of clinical symptoms and lethality of the disease. Thevaccine of the present disclosure is typically a genetically engineered(recombinant) mutant virus vaccine.

In the context of the present disclosure, the term “non-deficient in itsreplication” refers to a non-naturally occurring recombinant ASFV whichis able to replicate in vitro and/or in vivo and/or is capable ofproducing viral progeny although such replication and/or viral progenyproduction may also occur at reduced levels compared to the unmodifiedparent strain. Therefore, it can be the case that such ASFV is nondeficient in its replication in vitro, e.g. in a cell culture, althoughin vivo in a mammal such ASFV is at least partially impaired in itsreplication, e.g. resulting in a replication and/or viral progenyproduction below detection limits.

As used herein, the term “minimal dose” or “minimal effective dose”refers to a dose that demonstrates the absence of, or minimal presenceof, toxicity to the recipient, but still results in producing a desiredresult (e.g., protective immunity).

Viruses/Vaccines

Provided herein is a novel mutant ASFV-G ΔI1177L virus (SEQ ID NO: 2),resulting from the recombinant deletion of a portion of the I177L gene(SEQ ID NO: 3) of the parental ASFV-G genome (SEQ ID NO: 1). The genomicnucleotide sequence of a specific recombinant mutant ASFV-G ΔI177L (SEQID NO: 2) is described herein and differs from the genomic nucleotidesequence encoding the parental ASFV-G (SEQ ID NO: 1). The ASFV-GI177L-encoded protein of 177 amino acids (SEQ ID NO: 4) differs from thepredicted mutant I177L protein encoded by the mutant nucleotide sequenceof ASFV-G ΔI177L. The I177L protein (SEQ ID NO: 6) from ASFV-GΔI177L ispredicted to lack amino acids 112 through 150 of the wild-type I177Lprotein. Because the p72Mcherry Cassette is inserted in this position(see Examples section), it is not believed that the remaining codingregion after this insertion is transcribed, resulting in no functionalI177L protein being produced during viral infection.

The exemplary mutant strain (ASFV-G ΔI177L (SEQ ID NO: 2)) isrepresentative of the genus of recombinant vaccines in which the ASFVI177L gene is non-functional, which includes, without limitation,deletion mutants, nonsense mutants, insertional mutants, frameshiftmutants and other mutants resulting in non-expression of the I177Lprotein, or expression of a non-functional I177L protein. Otherrecombinant viruses envisioned include mutants in regulatory elementsresulting in non-expression or non-translation of the I177L protein.

Modifications intended to preclude functional expression of a targetprotein (e.g., I177L) or reduced expression or reduced activity of atarget protein can involve mutations of the DNA or gene encoding thetarget protein, including deletion of all or a portion of a target gene,including but not limited to the open reading frame of a target locus,transcriptional regulators such as promoters of a target locus, and anyother regulatory nucleic acid sequences positioned 5′ or 3′ from theopen reading frame, insertion of premature stop codons in the openreading frame, and insertions or deletions that shift the reading frameleading to premature termination of translation. Such deletionalmutations can be achieved using any technique known to those of skill inthe art. Reduced levels of the target protein or reduced activity of thetarget protein may also be achieved with point mutations or insertionsin the DNA or gene encoding the target protein. Mutational, insertional,and deletional variants of the disclosed nucleotide sequences and genescan be readily prepared by methods which are well known to those skilledin the art. Techniques used to achieve reduced levels and/or reducedactivity of the target protein may include CRISPR/Cas, TALEN, andZn-finger nuclease. It is well within the skill of a person trained inthis art to make mutational, insertional, and deletional mutations whichare equivalent in function to the specific ones disclosed herein.

The approaches described herein that were used to create a deletionmutant of I117L in ASFV-G can be used in different isolates of ASFV(such as isolates circulating in Asia, Europe or Africa), where afunctional I117L is present. Such approaches can be varied bymethodologies known in the art, such as using different selectionmarkers that can select recombinant virus by purification such as, butnot limited to, fluorescent proteins, enzymes such as beta-glucuronidaseor beta-galactosidase that can be used with chromogenic substrates, anddrug selection makers. Such approaches can also be used to create anymutation to the ORF of I177L as well as to regulatory elementscontrolling the expression and translation of the I177L gene thatresults in a non-functional I177L protein.

Mutants of I177L (and related strain-specific alleles) in other ASFVstrains and genotypes is also encompassed by the present disclosure.ASFV strains comprising synthetic mutations in nucleic acid sequencesthat exhibit at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ IDNO: 3 are encompassed in the instant invention. ASFV strains comprisingentire genomes with 95%, 96%, 97%, 98%, 99% or higher identity to SEQ IDNO: 2 are also encompassed in the instant invention.

The present disclosure further contemplates the combination of anon-functional I177L gene with other recombinant mutations. As such, itis not only wild-type viruses that can be modified as disclosed herein,but also strains containing non-naturally occurring mutations in othergenes or genomic regions (see, e.g., U.S. Pat. No. 9,814,771).

The present disclosure provides that such rationally-designed, live,attenuated ASFV-G ΔI177L can be incorporated into immunogeniccompositions to produce a vaccine effective to protect an animal, suchas a pig, from clinical ASF disease when challenged with ASFV-G. Thus,one object of the invention is to provide a method for protecting ananimal against ASFV-G by administering an effective amount of rationallydesigned live attenuated ASFV-G ΔI177L vaccine. In another embodiment,the present disclosure provides a method for eliciting a protectiveimmune response in an animal, preferably of the family Suidae (e.g.,domestic pigs (Sus scrofa domesticus), wild pigs (Sus scrofa scrofa),warthogs (Potamochoerus porcus), bushpigs (Potamochoerus larvatus),giant forest hogs (Hylochoerus meinertzhageni) as well as feral pigs),Such methods will typically comprise administering to such animal theone or more ASFV immunogenic compositions and vaccines described herein.

An additional object of the present disclosure is to provide a methodfor distinguishing animals infected with a wild-type ASFV from animalsvaccinated with a recombinant virus described herein. Such methodologiesfor differentiating infected from vaccinated animals (DIVA) can beaccomplished by serological tests that detect the difference betweenwild-type I177L protein and a mutant I177L protein. Alternately, suchmethodologies can include genetic screening approaches such as PCRamplification and detection of different products based. Typically, suchapproaches utilize one or more primer sets that flank the site of amutation and expand the same region, resulting in products of differentlengths or sequences.

The immunogenic composition(s) of the invention herein, regardless ofother components included, comprise a recombinant ASFV with anon-functional I177L gene/protein. I177L proteins of the presentinvention can comprise the entirety of SEQ ID NO: 4 and proteins with90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity tothe protein of SEQ ID NO: 4.

The immunogenically effective amounts of immunogenic compositionsdisclosed herein can vary based upon multiple parameters. In general,however, effective amounts per dosage unit for intramuscular applicationcan be about 10² 50% hemadsorption dose (“HAD₅₀”) to 10⁶ HAD₅₀. One,two, or more dosage units can be utilized in practicing themethodologies of the present invention. A dosage unit can readily bemodified to fit a desired volume or mass by one of skill in the art.Regardless of the dosage unit parameters, immunogenic compositionsdisclosed herein can be administered in an amount effective to producean immune response.

Dosage levels of active ingredients in vaccines disclosed herein, can bevaried by one of skill in the art to achieve a desired result in asubject or per application. As such, a selected dosage level can dependupon a variety of factors including, but not limited to, formulation,combination with other treatments, severity of a pre-existing condition,and the presence or absence of adjuvants. In preferred embodiments, aminimal dose of an immunogenic composition is administered.Determination of a minimal dose is well within the capabilities of oneskilled in the art.

Vaccines of the present invention can be prepared by conventionalmethods used for commercially available live attenuated ASFV vaccines.In a specific embodiment, a susceptible substrate is inoculated with aASFV-G ΔI177L mutant and propagated until the virus has replicated to adesired titer after which ASFV-G ΔI177L-containing material isharvested. Following this, the harvested material can be formulated intoa vaccine preparation with immunogenic properties. Every substrate whichis able to support the replication of the recombinant viruses providedherein can be used in the present invention, including primary culturesof swine peripheral blood macrophages or blood from infected swine.

Formulations and Administration

A vaccine provided herein comprises one of the recombinant viruses asdefined above in a live form, and a pharmaceutically acceptable carrieror diluent customarily used for such compositions. Carriers includestabilizers, preservatives and buffers. Suitable stabilizers include,for example, SPGA (sucrose, phosphate, glutamate and albumin),carbohydrates (sorbitol, mannitol, starch, sucrose, dextran, glutamate,and glucose), proteins (dried milk, serum, albumin, casein), ordegradation products thereof. Suitable buffers include, for examplealkali metal phosphates. Preservatives that can be utilized, include,but are not limited to, thimerosal, merthiolate and gentamicin. Diluentsinclude water, aqueous buffers (e.g., buffered saline), alcohols andpolyols (e.g., glycerol).

In some instances, vaccines of the present invention also contain orcomprise one or more adjuvants, which includes any material included inthe immunogenic composition formulation that enhances an immune responsein the recipient that is induced by the immunogenic composition. In someinstances, such adjuvants can include proteins other components includedwith the recombinant virus. Other adjuvants can be included as an extracomponent of the immunogenic compositions, and include such categoriesas aluminum salts (alum), oil emulsions, saponins, immune-stimulatingcomplexes (ISCOMs), liposomes, microparticles, nonionic blockcopolymers, derivatized polysaccharides, cytokines, and a wide varietyof bacterial derivatives. Any relevant adjuvant known in the art can beutilized in practicing the inventions disclosed herein. Factorsinfluencing the selection of an adjuvant include animal species,specific pathogen, antigen, route of immunization, and type of immunityneeded and can be readily determined by one of skill in the art.

Immunogenic compositions of the present disclosure can also comprisecarriers in addition to the recombinant virus. Carriers utilized inpracticing the immunogenic compositions provided herein can be any knownin the art and can be liquid, solid, semi-solid, or gel. The type offormulation can be modified depending on the route of administration ofthe antigen. Preferably, carriers are non-toxic to the recipient. One ofskill in the art is readily able to choose such carriers for applicationto recipient animals such as poultry.

The present disclosure provides immunogenic compositions for introducinga recombinant ASFV lacking a functional I177L gene/protein in acomposition containing, at a minimum, the recombinant virus, intotargets (e.g., swine). Thus, the compositions provided herein can beutilized to induce immunity or resistance to ASFV disease.

Vaccines provided herein may be administered by intramuscular,subcutaneous, intranasal or injection in an amount which is effective toprotect the animal against challenge by a virulent strain of ASFV. Thevaccine may be administered orally, through direct oral inoculation,dosed in drinking water, or though bait delivery systems. The effectiveamount of recombinant virus may vary according to parameters consideredby those skilled in the art. Effective amounts can be experimentallydetermined as necessary by those of skill in the art by following anyknown method or the guidance provided in the Examples herein.

Having generally described this invention, the same will be betterunderstood by reference to certain specific examples, which are includedherein to further illustrate the invention and are not intended to limitthe scope of the invention as defined by the claims.

EXAMPLES Example 1

Cell Culture and Viruses.

Primary swine macrophage cell cultures were prepared from defibrinatedswine blood as previously described (Zsak et al, J. Virol., (1998)72:1028-35). Briefly, heparin-treated swine blood was incubated at 37°C. for 1 hour to allow sedimentation of the erythrocyte fraction.Mononuclear leukocytes were separated by flotation over a Ficoll-Paque(Pharmacia, Piscataway, N.J.) density gradient (specific gravity,1.079). The monocyte/macrophage cell fraction was cultured in plasticPrimaria (Falcon; Becton Dickinson Labware, Franklin Lakes, N.J.) tissueculture flasks containing macrophage media, composed of RPMI 1640 Medium(Life Technologies, Grand Island, N.Y.) with 30% L929 supernatant and20% fetal bovine serum (HI-FBS, Thermo Scientific, Waltham, Mass.) for48 hours at 37° C. under 5% CO2. Adherent cells were detached from theplastic by using 10 mM EDTA in phosphate buffered saline (PBS) and werethen reseeded into Primaria T25, 6- or 96-well dishes at a density of5×10⁶ cells per ml for use in assays 24 hours later.

Virus titration was performed on primary swine macrophage cell culturesin 96-well plates. Virus dilutions and cultures were performed usingmacrophage medium. Presence of virus was assessed by hemadsorption (HA)and virus titers were calculated by the Reed and Muench method (Amer. J.Hygiene, (1938) 27:493-497).

ASFV Georgia (ASFV-G) utilized for this study was a field isolate kindlyprovided by Dr. Nino Vepkhvadze, from the Laboratory of the Ministry ofAgriculture (LMA) in Tbilisi, Republic of Georgia.

Example 2

Construction of a Recombinant ASFV-G ΔI177L

Recombinant ASFVs were generated by sequential homologous recombinationbetween the parental ASFV genome and recombination transfer vectors ininfection and transfection procedures using swine macrophage cellcultures (Neilan et al, Virol., (2004) 319:337-42; Zsak et al, supra).Recombinant transfer vector (p72GUSΔI177L) containing flanking genomicregions including portions of I177L mapping to the left (1 kbp) andright (1 kbp) of the gene and a reporter gene cassette containing themCherry gene with the ASFV p72 late gene promoter, p72mCherry was used.This construction created a 112-nucleotide deletion in the I177L ORF(amino acid residues 112 to 150) (FIG. 1). Recombinant transfer vectorp72mCherryΔI177L was obtained by DNA synthesis (Epoch Biosciences,Bothwell, Wash., USA). Macrophage cell cultures were infected withASFV-G and transfected with p72mCherryΔI177L. Recombinant virusesrepresenting independent primary plaques were purified to homogeneity bysuccessive rounds of plaque assay purification. The recombinant viruswas obtained after 14 successive plaque purification events onmonolayers of primary swine macrophage cell cultures.

Example 3

Full Genome Sequence Analysis of ASFV-G ΔI177L Relative to ParentalASFV-G.

To evaluate the accuracy of the genetic modification and the integrityof the genome of the recombinant virus, full genome sequences of ASFV-GΔI177L and parental ASFV-G were obtained using Next GenerationSequencing (NGS) and compared. As a first step, a full-length genomecomparison between the parental ASFV-G laboratory strain used toconstruct the ASFV-G ΔI177L mutant virus and the original ASFV Georgia2007/1 (Chapman et al, Emerg. Infect. Dis., (2001) 17:599-605; GenBankaccession FR682468) was performed. ASFV DNA was obtained from thecytoplasm of infected cells using the Trizol method (Life Technologies,Grand Island, N.Y., USA). DNA concentration was determined using theQubit® dsDNA HS assay kit (Life Technologies) and read on a Qubit® 2Flourometer (Life Technologies). In Brief, the viral DNA was shearedusing enzymatic reactions assessed for the distribution of sizefragmentation, then ligation of indentifying barcodes using an adaptersequence were added to the DNA fragments. Using a Pippin Prep™ (SageScience, Beverly, Mass.) the required size range of the library wascollected, and normalized. We then used this DNA library for NGSsequencing using the NextSeq (Illumnia, San Diego, Calif.) following themanufactures protocol. Sequence analysis was performed using CLCGenomics Workbench software (CLCBio, Waltham, Mass.).

The following differences were observed between these two viruses(nucleotide positions are provided based on ASFV Georgia 2007/1, GenBankaccession FR682468?): (i) three nucleotide insertions, T at position433, an A at position 441 in a non-coding segment of the genome, and a Aat position 174954 in the I177L gene, which causes I177L to merge withORF ASFV_G_ACD 01760, this additional nucleotide allows ORF I117L toresemble a similar full length gene as other isolates, as with theadditional A, there is not an early stop codon and out of frame mutationas described in the reference genome; (ii) two nucleotide deletions, Tat position 1602 and T at position 1603 in the MGF 360-1L gene ORFresulting in a frameshift; (iii) a nucleotide deletion, T at position1620 in the MGF 360-1L gene ORF resulting in a frameshift; (iv) anucleotide mutation, A to G at position 97391 resulting in a silentmutation in ORF B438L; (v) a nucleotide mutation, C to G at position166192 resulting in a residue substitution (Ala to Pro) at residueposition 85 in ORF E199L; and (vi) a nucleotide insertion, at position183303, a non-coding segment of the genome.

To determine if the recombinant virus acquired additional geneticchanges from the parent strain, a full-length genome comparison betweenASFV-G ΔI177L and the parental ASFV-G was performed. The DNA sequenceassemblies of ASFV-G ΔI177L and ASFV-G revealed a deletion of 112nucleotides in I177L gene corresponding with the introducedmodification. The consensus sequence of the ASFV-G ΔI1771 genome showedan insertion of 3944 nucleotides in I177L gene corresponding to thep72-mcherry cassette sequence introduced to generate a 112-nucleotidedeletion in the targeted gene. Besides the insertion of the cassette, noadditional differences were observed between ASFV-G ΔI1771 and ASFV-Ggenomes. In summary, ASFV-G ΔI1771 virus did not accumulate anysignificant mutations during the process of homologous recombination andplaque purification.

Example 4

Assessment of ASFV-G ΔI177L Virulence in Swine.

Animal experiments were performed under biosafety level 3 conditions inthe animal facilities at PIADC following a protocol approved by theInstitutional Animal Care and Use Committee.

ASFV-G ΔI177L was assessed for its virulence phenotype relative to thevirulent parental ASFV-G virus using 80-90-pound commercial breed swine.Five pigs were inoculated intramuscularly (IM) either with 10², 10⁴, 10⁶HAD₅₀ of ASFV-G ΔI177L or with 10² HAD₅₀ of ASFV-G virus. Clinical signs(anorexia, depression, fever, purple skin discoloration, staggeringgait, diarrhea and cough) and changes in body temperature were recordeddaily throughout the experiment. In protection experiments animals wereIM inoculated with 10², 10⁴, 10⁶ HAD₅₀ of ASFV-G ΔI177L and 28 dayslager IM challenged with 10² HAD₅₀ of the parental virulent ASFV-Georgia2007 strain. Presence of clinical signs associated with the disease wasassessed as described earlier.

All pigs inoculated via IM with 10⁴ HAD₅₀ of ASFV-G exhibited increasedbody temperature (>104° F.) by 3 to 4 days post-infection. Pigspresented clinical signs associated with the disease including anorexia,depression, purple skin discoloration, staggering gait and diarrhea(Table 1). Signs of the disease aggravated progressively over time andanimals either died or were euthanized in extremis by days 7 or 9post-infection. Conversely, animals inoculated via IM with either 10²,10⁴, or 10⁶ HAD₅₀ of ASFV-G ΔI177L did not present any signs of clinicaldisease during the entire observation period (21 days). Therefore,deletion of I177L gene produced a complete attenuation of the parentalvirulent ASFV-G. All animals in the Mock vaccinated group wereeuthanized due to humanitarian reasons following the corresponding IACUCprotocol.

TABLE 1 Swine survival and fever response following infection with 10²HAD₅₀ doses of ASFV-G-ΔI177L or parental ASFV-G. Fever Mean No. ofDuration time to days No. of Maximum No. of death to onset days dailytemp survivors/ (days ± (days ± (days ± (° F. ± Virus total SD) SD) SD)SD) ASFV-G 0/5 4.8 4.8 0.8 105.2 (0.84) (0.84) (0.84) (0.6)ASFV-G-ΔI177L 5/5 — — — 102.9 10²HAD₅₀ (0.5) ASFV-G-ΔI177L 5/5 — — —102.8 10⁴HAD₅₀ (0.57) ASFV-G-ΔI177L 5/5 — — — 102.8 10⁶HAD₅₀ (0.49)

Animals infected with ASFV-G presented with expected high homogenoustiters (10⁷⁵-10⁸⁵ HAD₅₀/ml) on day 4 pi, increasing (around 10⁸⁵HAD₅₀/ml) by day 7 pi when all animals were euthanized. Conversely,ASFV-G-ΔI177L revealed a different pattern with low viremia(10¹⁸-10^(2.3) HAD₅₀/ml) at day 4 pi, reaching peak values(10³⁸-10^(7.5) HAD₅₀/ml) by day 11 pi and then decreasing titers(10²³-10⁴HAD₅₀/ml) until day 28 pi (FIG. 3). It should be noted that oneof the five animals inoculated with ASFV-G-ΔI177L showed a remarkablylower viremia (1,000- to 10,000-fold lower depending on the time pointconsidered) than the average viremia values of the other animals in thegroup. Therefore, deletion of the I177L gene produced completeattenuation of the parental highly virulent ASFV-G virus when inoculatedat a low dose, with the infected animals presenting long viremias withrelatively low values.

Example 5

Protective Effect of ASFV-G ΔI177L Against Challenge with ParentalASFV-G.

Because pigs inoculated via IM with 10² HAD₅₀-10⁶ HAD₅₀ of ASFV-G ΔI177Lsurvived the infection without signs of the disease, groups of animals(n=5) inoculated with 10² HAD₅₀-10⁶ HAD₅₀ of ASFV-G ΔI177L werechallenged via IM with 10² HAD₅₀ of parental ASFV-G at day 28post-inoculation (homologous challenge). Five naive animals that werechallenged using the same route and dose served as anon-inoculated/challenged control group. All animals were IM vaccinatedwith 10² HAD₅₀ of ASFV-G ΔI177L and challenged IM 28 days later with 10²HAD₅₀ of ASFV-G virus. All animals in the Mock vaccinated group wereeuthanized due to humanitarian reasons following the corresponding IACUCprotocol. All animals in the ASFV-G ΔI177L vaccinated group remainclinically normal during the observational period of 21 days after thechallenge.

The five ASFV-G ΔI177L-inoculated and challenged animals remainedcompletely asymptomatic during all the observational period (21 days)(Table 2). All the animals in the mock inoculated/challenged controlgroup developed disease with a clinical course similar to that observedin animals inoculated with 10² HAD₅₀ of ASFV-G (see above). Therefore,ASFV-G ΔI177L is able to induce protection against the presentation ofclinical disease when challenged with the highly virulent parentalvirus.

TABLE 2 Swine survival and fever response in animals challenged withASFV-G virus at 28 days post-ASFV-G-ΔI177L infection. Fever Mean No. ofDuration time to days to No. of Maximum No. of death onset days dailytemp survivors/ (days ± (days ± (days ± (° F. ± Virus total SD) SD) SD)SD) Mock 0/5 5.6 4.2 1.4 105.6     (0.55)⁽¹⁾ (0.84) (0.88) (0.78)ASFV-G-ΔI177L 10/10 — — — 102.7 10²HAD₅₀ (0.68) ASFV-G-ΔI177L 5/5 — — —102.9 10⁴HAD₅₀ (0.37) ASFV-G-ΔI177L 5/5 — — — 103 10⁶HAD₅₀ (0.43)

Analysis of viremia in animals infected with ASFV-G presented withexpected high titers (10⁷³-10^(8.3) HAD₅₀/ml) on day 4 pi, increasing(averaging 10⁸⁵ HAD₅₀/ml) by day 7 pi when all animals were euthanized.After challenge, none of the ASFV-G-ΔI177L-infected animals had viremiaswith values higher than those present at challenge and viremia valuesdecreased progressively until the end of the experimental period (21days after challenge) when, importantly, no circulating virus could bedetected in blood from any of these animals (FIG. 5).

In summary, here we present evidence that deletion of the I177L genedrastically alters virulence of ASFV-G producing a completely attenuatedvirus named ASFV-G ΔI177L. Animals immunized with ASFV-G ΔI177L wereprotected against challenge with the virulent parental ASFV-G.

Example 6

The Ability of ASFV-G-I117L to Grow in Swine Macrophages

In vitro growth characteristics of ASFV-G-ΔI177L were evaluated inprimary swine macrophage cell cultures, the primary cell targeted byASFV during infection in swine and compared relative to parental ASFV-Gin multistep growth curves (FIG. 2). Cell cultures were infected at aMOI of 0.01 and samples were collected at 2, 24, 48, 72 and 96-hourspost-infection (hpi). Results demonstrated that ASFV-G-ΔI177L displayeda growth kinetic significantly decreased when compared to parentalASFV-G. ASFV-G-ΔI177L yields are approximately 100 to 1,000-fold lowerthan those of ASFV-G depending on the time point considered.

Therefore, deletion of the I177L gene significantly decreased theability of ASFV-G-ΔI177L, relative to the parental ASFV-G isolate, toreplicate in vitro in primary swine macrophage cell cultures.

Example 7

ASFV-G-ΔI117L Infected Animals do not Shed Vaccine Virus

In the above example where different groups of five pig were infected IMwith either 10², 10⁴, or 10⁶ HAD₅₀ of ASFV-G-ΔI177L, a mock infectedanimal was cohabitating in each of the groups as sentinel to detect thepotential virus shedding from the infected animals. All sentinel animalsremained clinically normal (FIG. 4). No virus was detected in any of thesamples obtained from sentinel animals (all sampled blood time points aswell as tonsil and spleen samples obtained at 28 days pi), indicatingthat ASFV-G-ΔI177L infected animals are not able to shed enough virus toinfect naive pigs for 28 days, a relatively long period of time ofcohabitation.

In summary, a non-vaccinated animal that comingled with vaccinatedanimals for 28 days did not present any clinical symptoms and allsampled blood, tonsil and spleen samples were negative for vaccinevirus, indicating that the vaccine virus was unable to shed to anon-vaccinated animal.

Example 8

Host Antibody Response in Animals Infected with ASFV-G-ΔI177L

All animals infected with ASFV-G-ΔI177L, regardless the dose of virusreceived possessed similar high titers of circulating anti-ASFVantibodies (FIG. 6). Antibody response, mediated by IgM and IgGisotypes, starts being detected in all three groups by day 12 pi. By day14 pi response mediated by both antibody isotypes reached maximum levelsin all groups. IgM mediated antibody response disappeared in all animalsby day 21 pi, while IgG mediated response stay high with minimalfluctuation until day 28 pi without significant differences betweenanimals in the three groups inoculated with ASFV-G-ΔI177L. Therefore,there is a close correlation between presence of anti-ASFV antibodies atthe moment of the challenge and protection. It should be mentioned thatno antibodies were detected in any serum sample obtained from thesentinel animals with the exception of the one sample at 28 days afterinfection with ASFV—in the group receiving 10⁶ HAD₅₀ of G-ΔI177L wherelow antibody titer was observed (FIG. 6).

Example 9

Induction of Sterile Immunity

Using an I177L specific real time PCR to specifically detect onlychallenge virus (which allows the detection of approximately 10 HAD₅₀)all blood samples tested negative for the presence of challenge virus.Furthermore, tonsils and spleen samples were obtained from all animalsat the end of the observational period (21 days post challenge) andtested for the presence of virus by virus isolation in swine macrophagecultures. Most of the animals in each group showed presence ofinfectious virus either in tonsils or spleen (data not shown). Allpositive samples were then assessed using the I177L specific real timePCR detecting the presence of the challenge virus in only one spleenbelonging to one of the animals initially infected with 10² HAD₅₀/ml ofASFV-G-ΔI177L. These results suggest that replication of challenge viruswas absent in all infected animals receiving 10⁴ HAD₅₀/ml or higher andmost of the animals receiving a 10² HAD₅₀/ml of ASFV-G-ΔI177L.

In summary, sterile immunity (immunity that doesn't allow thereplication of challenge virus) was achieved partially in animalsvaccinated with 10² HAD₅₀/ml of ASFV-G-ΔI177L and was fully achieved ata dose of 10⁴ HAD₅₀/ml or 10⁶ HAD₅₀/ml of ASFV-G-ΔI177L.

While the invention has been described with reference to details of theillustrated embodiments, these details are not intended to limit thescope of the invention as defined in the appended claims. Theembodiments of the disclosure in which exclusive property or privilegeis claimed is defined as follows.

What is claimed is:
 1. A genetically modified virus, wherein the virusgenome comprises a viral genome at least 95% identical to SEQ ID NO: 2.2. The virus of claim 1, wherein the virus genome comprises a viralgenome at least 99% identical to SEQ ID NO:
 2. 3. The virus of claim 1,wherein the viral genome comprises SEQ ID NO:2.
 4. A vaccine compositionagainst African Swine Fever Virus (ASFV), comprising the geneticallymodified virus of claim 1, wherein the ASFV is ASFV-Georgia 2007 isolate(ASFV-G).
 5. A method for the protection of swine against ASFV-G,comprising administering to a swine a live attenuated vaccine comprisingthe genetically modified virus of claim 1 in an amount effective toprotect said swine from clinical ASFV-G disease.
 6. A recombinant ASFVmutant virus, comprising a synthetic mutation in the I177L open readingframe or in a regulatory element controlling the expression of the I177Lprotein, resulting in a non-functional genomic I177L gene, wherein themutant ASFV comprises a genome at least 95% identical to SEQ ID NO: 2.7. The recombinant virus of claim 6, wherein the mutant ASFV comprises agenome at least 99% identical to SEQ ID NO: 2.